We will engage a multidisciplinary team from the University of California Lead Campus Program for Nanotoxicology in studying how the physicochemical characteristics of metal oxide (NP) influence biocompatibility and toxicity in vivo and in vitro. The long-term goal is to develop a rapid screening procedure that classifies NP into potentially safe or dangerous categories. Outline: Titanium dioxide (TiO2), zinc oxide (ZnO) and Ceria (CeO2/Ce2O3) metal oxide NP were chosen based on high volume of production, potential for airborne spread and ability to induce airway inflammation through the generation of reactive oxygen species (ROS). We are particularly interested in how a variation in the physicochemical characteristics of engineered NP influences their biocompatibility or toxicity in portal-of- entry (e.g., macrophages, epithelial, endothelial cells) cellular targets in the lung. In particular, we would like to determine whether our predictive hierarchical oxidative stress model, which is comprised of compensatory as well as injurious cellular responses, could be used for generating a high throughput screening procedure in tissue culture cells that can be used to predict the toxic potential of NP in vivo. To achieve this goal, we will determine how controlled design of the physicochemical characteristics (chemical composition, particle size, state of agglomeration, encapsulation, surface charge) of metal oxide influence ROS production in cells. Metal oxide NP will be synthesized by flame pyrollysis, followed by studying the NP characteristics under dry and wet conditions. We will determine how the variation in the design properties influences: (i) induction of incremental levels of oxidative stress that culminate in adaptive, pro-inflammatory and cytotoxic responses (Aim 1). (2) cellular uptake, subcellular localization and mitochondrial targeting as a prelude to cellular toxicity or biological adaptation (Aim 2); These in vitro cellular studies will involve the use of flow cytometry, real-time PCR, Western blotting, ELISA assays electron microscopy, and confocal microscopy. Once toxicity profiling has been accomplished, the different readouts will be combined into a high-throughput epifluorescence screening procedure that compares several particle types and design modifications simultaneously (Aim 3). Finally, we will determine how the variation in NP physicochemical characteristics influences the induction of airway inflammation in a murine intratracheal instillation model (Aim 4). We will also attempt to relate the vitro oxidant stress effects to the induction of in vivo oxidative stress by using a transgenic mouse that expresses the heme oxygenase 1 promoter linked to a luciferase reporter. We will determine whether the knockout of a key antioxidant defense regulator (Nrf2) renders these animals more susceptible to NP-induced oxidative stress injury. PUBLIC HEALTH RELEVANCE: This application will develop a novel testing strategy to screen for the safety of a large number of new nanomaterials that are coming onto the market. We will use the design of metal oxide nanoparticle as a screening tool to develop our toxicity screening method that will assess how these particles lead to cell death by different uptake mechanisms and ability to produce toxic oxygen radicals.